P847: BIOLOGICAL AND MOLECULAR CHARACTERIZATION OF IN SILICO IDENTIFIED PUTATIVE INHIBITORS OF PARASPECKLE ASSEMBLY WITH POTENTIAL ANTI-MULTIPLE MYELOMA ACTIVITY.
Authors: Valentina Traini, Giuseppina Fabbiano, Domenica Ronchetti, Ilaria Silvestris, Vanessa Katia Favasuli, Silvia Erratico, Domenica Giannandrea, Tommaso Laurenzi, Luca Palazzolo, Yvan Torrente, Raffaella Chiaramonte, Ivano Eberini, Niccolò Bolli, Antonino Neri, Elisa Taiana
Journal: HemaSphere (2023)

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: Paraspeckles (PSs) are membraneless nuclear bodies, assembled through the interaction between the lncRNA NEAT1 and PS proteins (PSPs), among which NONO. We previously highlighted that NEAT1 is significantly overexpressed in plasma cells of multiple myeloma (MM) patients and demonstrated that its silencing leads to PSs disassembly, negatively regulating proliferation and viability of MM cells. Accordingly, NEAT1 overexpression confers a pro-survival and pro-oncogenic advantage to MM cells exposed to stressful stimuli. Similarly, we recently reported higher NONO expression in primary CD138+ purified MM cells, correlating with poorer overall and progression free survival. Aims: On these bases we hypothesized that PSs could represent a novel specific vulnerability in MM. While clinical translation of lncRNA targeting may be problematic, protein components of the PS are more amenable to drug design. Here, we assess in vitro the on-target activity of the top 6 hit compounds resulting from an in silico screening of small molecules targeting crucial interaction points between NEAT1, NONO, and SFPQ and supposed to disrupt PSs structure, thus, at least in part, mimicking the NEAT1 silencing strategy. Methods: The biological activity of the 6 small molecules was evaluated in a panel of 6 MM cell lines (HMCLs), 4 haematological non-HMCLs and 6 healthy donors-derived PBMC samples. Dose-effect curves for the determination of IC50 values, were obtained by Trypan Blue exclusion and confirmed by CellTiter-Glo assay. Modulation of PSs integrity was analysed combining NEAT1 RNA-FISH and NONO immunofluorescence via confocal microscopy. Clonogenic potential was evaluated through methylcellulose assay. Cell cycle and apoptosis were investigated by FACS analysis. WB analysis was used to assess the levels of PSPs. Transcriptome analysis was performed by means of Clariom™ D arrays. Results: 2 of the 6 compounds demonstrated a significant reduction of the number of viable cells after 5 days of treatment in all the tested HMCLs but not in non-MM cells (≈75% vs. 25% of affected cellular fraction, respectively), confirming a specific anti-MM activity. Confocal microscopy showed a ≈45% decrease in the number of PSs per cell in treated HMCLs, validating the expected loss of PS integrity and the on-target activity of both molecules. In line with the reduced viability, cell cycle analysis of treated MM cells revealed a significant 2-fold increase of the cellular population in the Sub-G0/G1 phase, and a downregulation of the percentage of cells distributed in the S phase, in agreement with clonogenic potential results (median of 50 colonies for vehicle samples vs. 9 and 13 upon treatment with the 2 inhibitors). Flow cytometry analysis revealed a dose-dependent reduction of the percentage of viable cells and a concomitant increase of early and late apoptotic cells in treated HMCLs (2/5-fold increase, depending on the HMCL tested), suggesting a pro-apoptotic effect of both inhibitors on HMCLs. Furthermore, WB results highlighted a decrease of key PSPs levels after treatment, among which NONO and SFPQ. Transcriptomic analysis revealed the downregulation of several pathways associated with tumorigenic processes and the negative modulation of gene sets related to the circadian rhythm regulation, as expected. Summary/Conclusion: We identified 2 molecules that, by specifically targeting NEAT1 essential interactors, exert an anti-MM specific activity resulting in PS structural core impairment, cell cycle arrest and apoptosis, suggesting that PS targeting could represent a possible new druggable vulnerability in MM. Keywords: Multiple myeloma, Apoptosis, Targeted therapy